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Analysis of protein oligomeric species by sucrose gradients

Lookup NU author(s): Professor Tiago OuteiroORCiD


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© Springer Science+Business Media New York 2016. Protein misfolding, aggregation, and accumulation are a common hallmark in various neurodegenerative diseases. Invariably, the process of protein aggregation is associated with both a loss of the normal biological function of the protein and a gain of toxic function that ultimately leads to cell death. The precise origin of protein cytotoxicity is presently unclear but the predominant theory posits that smaller oligomeric species are more toxic than larger aggregated forms. While there is still no consensus on this subject, this is a central question that needs to be addressed in order to enable the design of novel and more effective therapeutic strategies. Accordingly, the development and utilization of approaches that allow the biochemical characterization of the formed oligomeric species in a given cellular or animal model will enable the correlation with cytotoxicity and other parameters of interest. Here, we provide a detailed description of a low-cost protocol for the analysis of protein oligomeric species from both yeast and mammalian cell lines models, based on their separation according to sedimentation velocity using high-speed centrifugation in sucrose gradients. This approach is an adaptation of existing protocols that enabled us to overcome existing technical issues and obtain reliable results that are instrumental for the characterization of the types of protein aggregates formed by different proteins of interest in the context of neurodegenerative disorders.

Publication metadata

Author(s): Tenreiro S, Macedo D, Marijanovic Z, Outeiro TF

Editor(s): Rune Matthiesen

Publication type: Book Chapter

Publication status: Published

Book Title: Proteostasis: Methods and Protocols

Year: 2016

Volume: 1449

Pages: 331-339

Online publication date: 10/09/2016

Acceptance date: 02/04/2016

Series Title: Methods in Molecular Biology

Publisher: Humana Press

Place Published: New York


DOI: 10.1007/978-1-4939-3756-1_21

Library holdings: Search Newcastle University Library for this item

ISBN: 9781493937561